Methods to manipulate quality attributes of polypeptides produced in cho cells

ABSTRACT

In accordance with the present invention, CHO cells expressing a recombinant polypeptide of interest are grown in media where the amino acids, vitamins, phosphate, lipids and/or antioxidant optimization is utilized to manipulate and/or control the protein quality attributes of the polypeptides. Polypeptides expressed in accordance with the present invention may be advantageously used in the preparation of pharmaceutical compositions.

This application is a divisional of U.S. patent application Ser. No.15/774,138, filed May 7, 2018, which is the National Stage Applicationfiled under 35 U.S.C. § 371 of PCT Application No. PCT/US2016/060782,filed Nov. 7, 2016, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/252,849, filed Nov. 9, 2015, under 35 U.S.C. §119(e). The entire teachings of the referenced application areincorporated herein by reference which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to Chinese Hamster Ovary (CHO) cellsexpressing a recombinant polypeptide of interest grown in media wherethe amino acids, vitamins, phosphate, lipids and/or antioxidantoptimization is utilized to manipulate and/or control the proteinquality attributes of the polypeptides. Polypeptides expressed inaccordance with the present invention may be advantageously used in thepreparation of pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Current ongoing efforts to maximize bioreactor productivity in both timeand volume directly affect the scale and capital investment required fora bioreactor suite. As cells reach higher concentrations more quickly,yield is increased; therefore, the number and scale of bioreactors canbe reduced. To that end, not only cell engineering, but also culturemedia and related chemical and physical environments are used to assistcells in reaching peak performance quickly, maintaining a high level aslong as possible while producing high quality protein.

Recombinantly produced protein products are increasingly becomingmedically and clinically important for use as therapeutics, treatmentsand prophylactics. Therefore, the development of reliable cell cultureprocesses that economically and efficiently achieve the desiredpolypeptide attributes is a desired and needed goal in the art.

SUMMARY OF THE INVENTION

The method of the invention is related to the optimization of cellculture media to control and/or mediated protein quality attributes ofrecombinant glycoprotein expressed in Chinese Hamster Ovary (CHO) cellsby changing the level of one or more cell culture media componentsincluding but not limit to: growth factors, phosphate, Zn, Fe, aminoacids (Asp, Asn, Cys, Ser), lipids, vitamins (B1, B2, B3, B9, B12, Bx),antioxidants (ascorbic acid, glutathione, reduced a-lipoic acid, vitaminE), and combinations thereof.

One embodiment of the invention is controlling protein qualityattributes of a recombinant glycoprotein expressed in CHO cellscomprising modulating the amount of one or more cell culture mediacomponents, such as asparagine, aspartic acid, zinc and iron, whereinglycoprotein aggregation is decreased.

Another embodiment of the invention is controlling protein qualityattributes of a recombinant glycoprotein expressed in CHO cellscomprising modulating the amount of one or more cell culture mediacomponents, such as aspartic acid, phosphate and zinc wherein the sialicacid content of the glycoprotein is increased.

One embodiment of the invention is controlling protein qualityattributes of a recombinant glycoprotein expressed in CHO cellscomprising modulating the amount of one or more cell culture mediacomponents, such as asparagine, zinc and iron, wherein the galactosecontent of the glycoprotein is increased.

In one embodiment of the invention, the cell culture media is chemicallydefined. In another embodiment of the invention the cell culture mediabeing optimized is a basal medium.

In one embodiment of the invention, the cell culture medium does notcontain added serum or hydrolysates and/or may be protein-free.

The glycoprotein of the invention are recombinant antibodies, antibodyfragments or fusion proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the experimental and analytical flow chart. ProA: proteinA.

FIG. 2 shows clone A, clone B and clone G cells cultured in the presenceof growth factor (1 mg/L insulin) resulted in higher HMW % compared tothe cultures without. GF: growth factor; GF-Free: growth factor free.

FIGS. 3A-3D show the statistical analysis of HMW % for clone A. FIG. 3A:the levels of HMW % for proteins produced from cells cultured in theprototype media.

FIG. 3B: statistical summary for the mathematical model generated to fitthe data shown in 3A. FIG. 3C: List of the major effects of the factorson HMW % and the terms incorporated into the mathematical model. FIG.3D: predicted maximum or minimum HMW % levels using the model generated.

FIGS. 4A-4D show the statistical analysis of HMW % for clone C. FIG. 4A:the levels of HMW % for proteins produced from cells cultured in theprototype media.

FIG. 4B: statistical summary for the mathematical model generated to fitthe data shown in 4A. FIG. 4C: List of the major effects of the factorson HMW % and the terms incorporated into the mathematical model. FIG.4D: predicted maximum or minimum HMW % levels using the model generated.

FIGS. 5A-5D show the statistical analysis of HMW % for clone D. FIG. 5A:the levels of HMW % for proteins produced from cells cultured in theprototype media. FIG. 5B: statistical summary for the mathematical modelgenerated to fit the data shown in 5A. FIG. 5C: List of the majoreffects of the factors on HMW % and the terms incorporated into themathematical model. FIG. 5D: predicted maximum or minimum HMW % levelsusing the model generated.

FIGS. 6A-6D show the statistical analysis of HMW % for clone A. FIG. 6A:the levels of HMW % for proteins produced from cells cultured in theprototype media. FIG. 6B: statistical summary for the mathematical modelgenerated to fit the data shown in 6A. FIG. 6C: List of the majoreffects of the factors on HMW % and the terms incorporated into themathematical model. FIG. 6D: predicted maximum or minimum HMW % levelsusing the model generated.

FIGS. 7A-7D show the statistical analysis of BMW % for clone B. FIG. 7A:the levels of BMW % for proteins produced from cells cultured in theprototype media. FIG. 7B: statistical summary for the mathematical modelgenerated to fit the data shown in 7A. FIG. 7C: List of the majoreffects of the factors on HMW % and the terms incorporated into themathematical model. FIG. 7D: predicted maximum or minimum HMW % levelsusing the model generated.

FIG. 8 shows clone A, clone B and clone D cells cultured in the presenceof growth factor (1 mg/L insulin) resulted in lower gal % compared tothe cultures without. GF: growth factor; GF-Free: growth factor free.

FIGS. 9A-9D show the statistical analysis of gal % for clone A. FIG. 9A:the levels of gal % for proteins produced from cells cultured in theprototype. FIG. 9B: statistical summary for the mathematical modelgenerated to fit the data shown in 9A. FIG. 9C: List of the majoreffects of the factors on gal % and the terms incorporated into themathematical model. FIG. 9D: predicted maximum or minimum gal % levelsusing the model generated.

FIGS. 10A-10D show the statistical analysis of gal % for clone B. FIG.10A: the levels of gal % for proteins produced from cells cultured inthe prototype media. FIG. 10B: statistical summary for the mathematicalmodel generated to fit the data shown in 10A. FIG. 10C: List of themajor effects of the factors on gal % and the terms incorporated intothe mathematical model. FIG. 10D: predicted maximum or minimum gal %levels using the model generated.

FIGS. 11A-11D: Statistical Analysis of gal % for clone C. FIG. 11A: thelevels of gal % for proteins produced from cells cultured in theprototype media. FIG. 11B: statistical summary for the mathematicalmodel generated to fit the data shown in 11A. FIG. 11C: List of themajor effects of the factors on gal % and the terms incorporated intothe mathematical model. FIG. 11D: predicted maximum or minimum gal %levels using the model generated.

FIGS. 12A-12D show the statistical analysis of gal % for clone D. FIG.12A: the levels of gal % for proteins produced from cells cultured inthe prototype media. FIG. 12B: statistical summary for the mathematicalmodel generated to fit the data shown in A. FIG. 12C: List of the majoreffects of the factors on gal % and the terms incorporated into themathematical model. FIG. 12D: predicted maximum or minimum gal % levelsusing the model generated.

FIGS. 13A-13D show the statistical analysis of gal % for clone A. FIG.13A: the levels of gal % for proteins produced from cells cultured inthe prototype media. FIG. 13B: statistical summary for the mathematicalmodel generated to fit the data shown in 13A. FIG. 13C: List of themajor effects of the factors on gal % and the terms incorporated intothe mathematical model. FIG. 13D: predicted maximum or minimum gal %levels using the model generated.

FIGS. 14A-14D show the statistical analysis of gal % for clone F. FIG.14A: the levels of gal % for proteins produced from cells cultured inthe prototype media. FIG. 14B: statistical summary for the mathematicalmodel generated to fit the data shown in 14A. FIG. 14C: List of themajor effects of the factors on gal % and the terms incorporated intothe mathematical model. FIG. 14D: predicted maximum or minimum gal %levels using the model generated.

FIG. 15 shows the effect of growth factors and phosphate on gal % forclone B. Cells were cultured in the presence or absence of growthfactors with varied concentration of phosphate. Note that gal % forGF-Free conditions were higher than that for GF conditions regardless ofthe phosphate concentration. Phosphate decreased the gal % for either GFor GF-free conditions. GF: growth factor; GF-free: growth factor free.

FIG. 16 shows the effect of growth factors and phosphate on gal % forclone G. Cells were cultured in the presence or absence of growthfactors with varied concentration of phosphate. Note that gal % forGF-Free conditions were higher than that for GF conditions whenphosphate concentration was below 1.1 g/L. Increase Phosphate from 0.5to 1.1 g/L elevated the gal % for GF-free conditions; similarly,increase phosphate from 0.5 to 1.4 g/L led to higher gal % for GFconditions. GF: growth factor; GF-free: growth factor free.

FIG. 17 shows clone B cultured in the presence of growth factor (1 mg/Linsulin) resulted in lower SA % compared to the cultures without. GF:growth factor; GF-Free: growth factor free.

FIGS. 18A-18D show the statistical analysis of SA % for clone B. FIG.18A: the levels of SA % for proteins produced from cells cultured in theprototype media. FIG. 18B: statistical summary for the mathematicalmodel generated to fit the data shown in 18A. FIG. 18C: List of themajor effects of the factors on SA % and the terms incorporated into themathematical model. FIG. 18D: predicted maximum or minimum SA % levelsusing the model generated.

FIG. 19 shows the effect of growth factors and phosphate on SA % forclone B. Cells were cultured in the presence or absence of growthfactors with varied concentration of phosphate. Note that SA % forGF-Free conditions were higher than that for GF conditions regardless ofthe phosphate concentration. Phosphate decreased the SA % for both GFand GF-free conditions. GF: growth factor; GF-free: growth factor free.

FIGS. 20A-20D show the statistical analysis of SA % for clone B. FIG.20A: the levels of SA % for proteins produced from cells cultured in theprototype media. FIG. 20B: statistical summary for the mathematicalmodel generated to fit the data shown in 20A. FIG. 20C: List of themajor effects of the factors on SA % and the terms incorporated into themathematical model. FIG. 20D: predicted maximum or minimum SA % levelsusing the model generated

FIGS. 21A-21D show the statistical analysis of man5% for clone G. FIG.21A: the levels of man5% for proteins produced from cells cultured inthe prototype media. FIG. 21B: statistical summary for the mathematicalmodel generated to fit the data shown in 21A. FIG. 21C: List of themajor effects of the factors on man5% and the terms incorporated intothe mathematical model. FIG. 21D: predicted maximum or minimum man5%levels using the model generated.

FIG. 22 shows the effect of growth factors and phosphate on man5% forclone G. Cells were cultured in the presence or absence of growthfactors with varied concentration of phosphate. Note that man5% forGF-Free conditions were lower than that for GF conditions regardless ofthe phosphate concentration. Phosphate decreased the man5% for both GFand GF-free conditions. GF: growth factor; GF-free: growth factor free.

FIG. 23 shows that growth factor (1 mg/L insulin) decreased man5% forclone D. GF: growth factor; GF-free: growth factor free.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new methods to manipulate and/orcontrol the protein quality attributes of polypeptides produced inChinese Hamster Ovary (CHO) cell cultures by changing the level ofspecific medium components including but not limit to: growth factors,phosphate, Zn, Fe, amino acids (Asp, Asn, Cys, Ser), lipids, vitamins(B1, B2, B3, B9, B12, Bx), and combinations thereof. As describedherein, a variety of media are encompassed by the present invention.

As used herein, the term “basal medium” and “basal media” refers tostarting medium to which cells are added to begin the culture. Basalmedia is a solution containing nutrients that nourish growing cells.Typically, a basal medium provides essential and non-essential aminoacids, vitamins, energy sources, lipids, and trace elements required bythe cell for minimal growth and/or survival. A basal medium may alsocontain supplementary components that enhance growth and/or survivalabove the minimal rate, including, but not limited to, hormones and/orother growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, and/or glucose or otherenergy source. In certain embodiments, a basal medium is advantageouslyformulated to manipulate and/or maintain recombinant polypeptide qualityattributes, such as percent high molecular weight (HMW %), percentgalactose (Gal %), percent sialic acid (SA %), and percent mannose (Man%) Exemplary basal media components are shown in Table 1.

As used herein, the term “amino acid” refers to any of the twentynaturally occurring amino acids that are normally used in the formationof polypeptides, analogs or derivatives of those amino acids or anynon-naturally occurring amino acid. In certain embodiments, amino acidsof the present invention are provided in medium to cell cultures. Theamino acids provided in the medium may be provided as salts or inhydrate form.

As used herein the term “defined medium” refers to a medium in which thecomposition of the medium is both known and controlled. In certainembodiments, basal media is a defined medium.

As used herein the term “glycoprotein” refers to a protein orpolypeptide that contains one or more covalently linked oligosaccharidechains. The oligosaccharide chains may be composed of a single sugarresidue, a single unbranched chain of sugar residues or a chain of sugarresidues that branches one or more times. The oligosaccharide chains maybe either N-linked or O-linked.

As used herein the term “glycosylation pattern” refers to the observedglycosylation of a given glycoprotein or glycoproteins. A glycoproteinwith a greater number of covalently linked sugar residues in itsoligosaccharide chain(s) is said to have an increased or more extensiveglycosylation pattern. Conversely, a glycoprotein with fewer covalentlylinked sugar residues in its oligosaccharide chain(s) is said to have adecreased or less extensive glycosylation pattern. The term“glycosylation pattern” as used herein also refers to a characteristicdistribution of several different glycosylation patterns on individualglycoproteins expressed according to the teachings of the presentinvention. In this sense, an increased glycosylation pattern refers toan increase in the characteristic distribution of glycosylation patternsof the expressed glycoproteins.

As used herein “recombinantly expressed polypeptide” and recombinantpolypeptide” refer to a polypeptide expressed from a host cell that hasbeen manipulated by the hand of man to express that polypeptide. Incertain embodiments, the host cell is Chinese Hamster Ovary cell. Incertain embodiments, this manipulation may comprise one or more geneticmodifications. For example, the host cells may be genetically modifiedby the introduction of one or more heterologous genes encoding thepolypeptide to be expressed. The heterologous recombinantly expressedpolypeptide can be identical or similar to polypeptides that arenormally expressed in the host cell. The heterologous recombinantlyexpressed polypeptide can also be foreign to the host cell, e.g.heterologous to polypeptides normally expressed in the host cell. Incertain embodiments, the heterologous recombinantly expressedpolypeptide is chimeric. For example, portions of a polypeptide maycontain amino acid sequences that are identical or similar topolypeptides normally expressed in the host cell, while other portionscontain amino acid sequences that are foreign to the host cell.Additionally or alternatively, a polypeptide may contain amino acidsequences from two or more different polypeptides that are both normallyexpressed in the host cell. Furthermore, a polypeptide may contain aminoacid sequences from two or more polypeptides that are both foreign tothe host cell. In some embodiments, the host cell is geneticallymodified by the activation or upregulation of one or more endogenousgenes. In certain embodiments, the recombinantly expressed polypeptideis an antibody. In certain embodiments, the recombinantly expressedpolypeptide is an antibody fragment (dAb).

Screening of Media Components

The media components were grouped into amino acids, vitamins, lipids,antioxidants, and phosphate based on the functions or properties for thechemical compounds. The groups were then the study factors used togenerate prototype media conditions shown in Example 2 using a responsesurface statistical design approach. Cells were cultured in theprototype media without any growth factors following the protocoldescribed in Example 1.

TABLE 1 Grouped components, levels and concentration range thereof(Example 2) Level 1 Level 2 Level 3 Concentration Group Components(Coded) (Coded) (Coded) Range Amino L-Aspartic Acid 0 (−1) +250% (0)+500% (+1) 0.1-2 g/L Acids L-Cysteine 0 (−1) +50% (0) +100% (+1) 0.1-2g/L L-Serine 0 (−1) +25% (0) +50% (+1) 0.1-2 g/L Vitamins Niacinamide(B3) 0 (−1) +25% (0) +50% (+1) 0.01-100 mg/L p-Aminobenzoic 0 (−1) +25%(0) +50% (+1) 0.01-100 mg/L Acid (Bx, PABA) Riboflavin (B2) 0 (−1) +25%(0) +50% (+1) 0.01-100 mg/L Thamine (B1) 0 (−1) +50% (0) +100% (+1)0.01-100 mg/L Phosphate Sodium Phosphate 0 (−1) +50% (0) +100% (+1)0.1-2 g/L Lipids Phosphotydylcholine Proprietary Proprietary ProprietaryAntioxidants Ascorbic Acid 0 (−1) +75% (0) +150% (+1) 0.01-100 mg/LGlutathione, reduced 0 (−1) +200% (0) +400% (+1) 0.01-100 mg/L a-LipoicAcid 0 (−1) +50% (0) +100% (+1) 0.01-100 mg/L Vitamin E 0 (−1) +50% (0)+100% (+1) 0.01-100 mg/L

Factors Impacting BMW %

Aggregation is a concern in biological manufacturing inasmuch asaggregated protein in the final product may affect biological activityand has been linked to the development of adverse immunologicalresponses (Rosenberg A S. Effects of Protein Aggregates: An ImmunologicPerspective. AAPS J 2006; 8 (3) E501-7).

Table 2 below summarizes the effects of a single factor on percent highmolecular weight (HMW %) across the different clones.

TABLE 2 Effect of Single Factor on HMW % Amino Growth Clone AcidsPhosphate Lipids Antioxidants Vitamins FeSO4 Zn Asn Asp Factors A NE NENE NE NE ↑ ↓ NE ↓ ↑ B NM NM NM NM NM NE ↑ NE ↑ C ↓ NE NE NE NE NM NM NMNM NM D ↑ ↑ ↓ ↑ ↓ NM NM NM NM NM G NM NM NM NM NM NM NM NM NM ↑ NE: NoEffect NM: No Measurement

While the addition of growth factors generally increased (↑) the HMW %,the addition of zinc generally decreased (↓) HMW %. In clone D, theaddition of amino acids, phosphate or antioxidants increased HMW % andthe addition of lipids or vitamins decreased HMW %.

Table 3 below summarizes the combined factors that have an effect on HMW% in three of the studied clones. The predictions are based on relativeranges: −1 is the lower range, 0 is the middle, and +1 is the higherrange. All of the ranges are relative to the components in the mediaformulation.

TABLE 3 Effect of Combined Factors on HMW % Concentration LevelsPrediction HMW % Amino Clone Target Level [Range] Acids Phosphate LipidsAntioxidants Vitamins A Max 7.53 [6.95, 8.11] −1 1 0.66 −1 1 Min 4.72[4.26, 5.18] 0.24 1 1 1 1 C Max 5.88 [5.34, 6.42] −1 1 N/A N/A N/A Min4.56 [4.07, 5.04] 1 −0.1 N/A N/A N/A D Max  10.56 [10.10, 11.03] 0.45 1−1 −0.31 −1  Min 3.85 [3.17, 4.52] 1 1 1 −1 1

In the case of clone A, the HMW % level could be minimized at a meanvalue of 4.72% with a 95% chance the HMW % will be in the range of 4.26to 5.18. However, if the levels for amino acids, lipids, or antioxidantsare not properly set, the HMW % could increase to as high as 8.11%.

In the case of clone C, the HMW % level could be minimized at a meanvalue of 4.56% with a 95% chance the HMW % will be in the range of 4.07to 5.04. However, if the levels for amino acids or phosphate are notproperly set, the HMW % could increase to as high as 6.42%.

In the case of clone D, the HMW % level could be minimized at a meanvalue of 3.85% with a 95% chance the HMW % will be in the range of 3.17to 4.52. However, if the levels for amino acids lipids, antioxidants orvitamin are not properly set, the HMW % could increase to as high as11.03%.

Table 4 below summarizes the effects specific ingredients have on HMW %in clone A and B.

TABLE 4 Effect of Combined Factors on HMW % Concentration LevelsPrediction HMW % Iron Clone Target Level [Range] Zn Asn FeSO4 CarrierAsp A Max 8.48 [8.00, 8.95] 0 0.14 1 IC1 1 Min 3.13 [2.87, 3.39] 1 1 1SC −1 B Max 6.48 [5.40, 7.54] 0 1 −1 N/A 1 Min 1.39 [0.00, 2.81] 1 10.04 N/A −1

In the case of clone A, the HMW % level could be minimized at a meanvalue of 3.13% when zinc, Asn and Asp were controlled at 1, 1, −1,respectively, with a 95% chance the HMW % will be in the range of 2.87to 3.39. However, if the levels for Asn, Asp or zinc are not properlyset, the HMW % could increase to as high as 8.95%.

In the case of clone B, the HMW % level could be minimized at a meanvalue of 1.39% when zinc, FeSO4 and Asp were controlled at 1, 0.04, −1,respectively, with a 95% chance the HMW % will be in the range of 0 to2.81. However, if the levels for Asp, zinc or FeSO4 are not properlyset, the HMW % could increase to as high as 7.54%.

Glycosylation

Glycosylation is the most common post-translational modification ofproteins. It is a complex process involving many functional proteins andresulting in a great diversity of carbohydrate-protein bonds and glycanstructures. Glycosylation of some proteins has a great impact on theirstructures, functions, stability and serum clearance rates, which allimpact efficacy.

Structurally, glycoproteins consist of a polypeptide covalently bondedto a carbohydrate moiety. The carbohydrate can make up anywhere fromless than one percent to more than 80 percent of the total protein mass.The saccharide chains, referred to as glycans, can be linked to thepolypeptide in two major ways. The first class of glycoproteins are the0-linked glycans. These usually contain an N-acetylgalactosamine whichis attached through a glycosidic bond to the 0-terminus of eitherthreonine or serine. The other class of glycoproteins are the N-linkedglycans. These involve a glycosidic bond between N-acetylglucosamine andthe N-terminus of an asparagine residue (Schulz, Georg E. And R. H.Schirmer. Principles of Protein Structure. Springer-Verlag: New York,1979. P. 228-230).

Attached to the N-acetylgalactosamine or N-acetylglucoseamine is one ormore mannose, and galactose residues, with a sialic acid residueoccupying the terminal positions of the oligosaccharide chains.

Factors Impacting Gal %

Table 5 below summarizes the effects of a single factor on percentgalactose (Gal %) across the different clones.

TABLE 5 Effect of Single Factor on Gal % Amino Growth Clone AcidsPhosphate Lipids Antioxidants Vitamins FeSO4 Zn Asn Factors A ↓ ↑ NE NENE ↓ ↑ ↓ ↓ B ↓ ↓ NE NE NE NM NM NM ↓ C ↓ NE NE NE NE NM NM NM NM D NE ↑↓ ↑ ↑ NM NM NM ↓ F NM NM NM NM NM ↑ NE ↓ NM G NM ↑ NM NM NM NM NM NM ↓NE: No Effect NM: no measurement

While the addition of amino acids, specifically Asn or growth factorsgenerally decreased (↓) the Gal %, the addition of zinc, vitamins andantioxidants generally increased (↑) Gal %. In clone D, the addition ofphosphate, vitamins or antioxidants increased Gal % and the addition oflipids or growth factors decreased Gal %.

Table 6 below summarizes the combined factors that have an effect on Gal% in clones A-D. The predictions are based on relative ranges: −1 is thelower range, 0 is the middle, and +1 is the higher range. All of theranges are relative to the components in the media formulation.

TABLE 6 Effect of Combined Factors on Gal % Concentration LevelsPrediction Gal % Amino Clone Target Level [Range] Acids Phosphate LipidsAntioxidants Vitamins A Max 34.15 [31.34, 36.96] −1 1 1 1 1 Min 9.27[6.26, 12.28] 1 −1 −1 1 1 B Max 116.77 [113.22, 120.33] −1 −1 Min 106.75[103.26, 110.24] 1 1 C Max 17.74 [16.19, 19.29] −1 −1 1 −1 Min 9.94[8.50, 11.37] 1 1 1 −1 D Max 38.56 [36.75, 40.37] −1 1 −1 1 1 Min 13.80[12.63, 14.97] −1 1 1 −1 −1

In the case of clone A, the Gal % level could be maximized at a meanvalue of 34.14% with a 95% chance the Gal % will be in the range of31.34 to 36.96%. However, if the levels for amino acids, phosphate orlipids are not properly set, the Gal % could decrease to as low as6.26%.

In the case of clone B, the Gal % level could be maximized at a meanvalue of 116.77% with a 95% chance the Gal % will be in the range of113.22 to 120.33%. However, if the levels for amino acids or phosphateare not properly set, the Gal % could decrease to as low as 103.26%.

In the case of clone C, the Gal % level could be maximized at a meanvalue of 17.74% with a 95% chance the Gal % will be in the range of16.19 to 1929%. However, if the levels for amino acids or lipids are notproperly set, the Gal % could decrease to as low as 8.50%.

In the case of clone D, the Gal % level could be maximized at a meanvalue of 38.56% with a 95% chance the Gal % will be in the range of36.75 to 40.37%. However, if the levels for lipids, antioxidants orvitamins are not properly set, the Gal % could decrease to as low as12.63%.

Table 7 below summarizes the effects specific ingredients have on Gal %in clone A, B and F.

FIG. 7 Effect of Combined Factors on Gal %

Concentration Levels Prediction Gal % Iron Clone Target Level [Range] ZnAsn FeSO4 Carrier Asp A Max 22.82 [20.51, 25.13] 1 −1 −1 SC Min 6.97[4.62, 9.32] 0 1 1 None B Max 115.60 [103.91, 127.29] 1 SC Min 74.75[63.06, 86.44] 1 E F Max 20.76 [19.64, 21.89] −1 1 Min 9.97 [8.56,11.38] 1 −1

In the case of clone A, the Gal % level could be maximized at a meanvalue of 22.82% when zinc, Asn and FeSO4 were controlled at 1, −1, −1,respectively, with a 95% chance the Gal % will be in the range of 20.51to 25.13%. However, if the levels for zinc, Asn, and FeSO4 are notproperly set, the Gal % could decrease to as low as 4.62%.

In the case of clone F, the Gal % level could be maximized at a meanvalue of 20.76% when Asn and FeSO4 were controlled at −1, 1,respectively, with a 95% chance the Gal % will be in the range of 19.64to 21.98. However, if the levels for Asn and FeSO4 are not properly set,the Gal % could decrease to as low as 8.562%.

Factors Impacting SA %

Table 8 below summarizes the effects of a single factor on percentsialic acid (SA %) in clone B.

TABLE 8 Effect of Single Factor on SA % Amino Growth Clone AcidsPhosphate Lipids Antioxidants Vitamins FeSO4 Zn Asn Asp Factors B ↑ NENE NE NE ↑ NE NE: No Effect

While the addition of amino acids and zinc generally increased (↑) theSA %, the addition of phosphate, Asp and growth factors generallydecreased (↓) SA %.

Table 9 below summarizes the combined factors that have an effect on SA% in clone B. The predictions are based on relative ranges: −1 is thelower range, 0 is the middle, and +1 is the higher range. All of theranges are relative to the components in the media formulation.

TABLE 9 Effect of Combined Factors on SA % Concentration LevelsPrediction SA % Amino Clone Target Level [Range] Acids Phosphate LipidsAntioxidants Vitamins B Max 36.74 [35.70, 37.78] 0.63 −1 Min 29.63[28.36, 30.90] −1 1

In the case of clone B, the SA % level could be maximized at a meanvalue of 36.74% with a 95% chance the SA % will be in the range of 35.70to 37.78%. However, if the levels for amino acids or phosphate are notproperly set, the SA % could decrease to as low as 28.36%.

Table 10 below summarizes the effects specific ingredients have on SA %in clone B.

TABLE 10 Effect of Combined Factors on SA % Concentration Levels IronPrediction SA % Car- Clone Target Level [Range] Zn Asn FeSO4 rier Asp BMax 47.39 [42.55, 52.23] 1 1 SC −1 Min 9.65 [6.03, 13.27] 1 1 E 1

In the case of clone B, the SA % level could be maximized at a meanvalue of 47.39% when Asp was controlled at −1, with a 95% chance the SA% will be in the range of 42.55 to 52.23%. However, if the level of Aspis not properly set, the SA % could decrease to as low as 6.03%.

Factors Impacting Man5%

Table 11 below summarizes the effects of a single factor on percentmannose 5 (Man5%) in clones D and G. While the addition of asparagine orgrowth factors generally increased (↑) the Man5%, the addition ofphosphate or zinc generally decreased (↓) Man5%.

TABLE 11 Effect of Single Factor on Man5% Amino Growth Clone AcidsPhosphate Lipids Antioxidants Vitamins FeSO4 Zn Asn Factors D NE NE NENE NE NE NE NE ↑ G NE ↓ NE NE NE NE ↓ ↑ ↑ NE: No Effect

Cells, Proteins and Cell Culture

In the cell culture processes or methods of this invention, the cellscan be maintained in a variety of cell culture media. i.e., basalculture media, as conventionally known in the art. For example, themethods are applicable for use with large volumes of cells maintained incell culture medium, which can be supplemented with nutrients and thelike. Typically, “cell culturing medium” (also called “culture medium”)is a term that is understood by the practitioner in the art and is knownto refer to a nutrient solution in which cells, preferably animal ormammalian cells, are grown and which generally provides at least one ormore components from the following: an energy source (usually in theform of a carbohydrate such as glucose); all essential amino acids, andgenerally the twenty basic amino acids, plus cysteine; vitamins and/orother organic compounds typically required at low concentrations; lipidsor free fatty acids, e.g., linoleic acid; and trace elements, e.g.,inorganic compounds or naturally occurring elements that are typicallyrequired at very low concentrations, usually in the micromolar range.Cell culture medium can also be supplemented to contain a variety ofoptional components, such as hormones and other growth factors, e.g.,insulin, transferrin, epidermal growth factor, serum, and the like;salts, e.g., calcium, magnesium and phosphate, and buffers, e.g., HEPES;nucleosides and bases, e.g., adenosine, thymidine, hypoxanthine; andprotein and tissue hydrolysates, e.g., hydrolyzed animal protein(peptone or peptone mixtures, which can be obtained from animalbyproducts, purified gelatin or plant material); antibiotics, e.g.,gentamycin; and cell protective agents, e.g., a Pluronic polyol(Pluronic F68). Preferred is a cell nutrition medium that is serum-freeand free of products or ingredients of animal origin, and chemicallydefined.

As is appreciated by the practitioner, animal or mammalian cells arecultured in a medium suitable for the particular cells being culturedand which can be determined by the person of skill in the art withoutundue experimentation. Commercially available media can be utilized andinclude, for example, Minimal Essential Medium (MEM, Sigma, St. Louis,Mo.); Ham's F10 Medium (Sigma); Dulbecco's Modified Eagles Medium (DMEM,Sigma); RPMI-1640 Medium (Sigma); HyClone cell culture medium (HyClone,Logan, Utah); and chemically-defined (CD) media, which are formulatedfor particular cell types. To the foregoing, exemplary media can beadded the above-described supplementary components or ingredients,including optional components, in appropriate concentrations or amounts,as necessary or desired, and as would be known and practiced by thosehaving in the art using routine skill.

In addition, cell culture conditions suitable for the methods of thepresent invention are those that are typically employed and known forbatch, fed-batch, or continuous culturing of cells, with attention paidto pH (e.g., about 6.5 to about 7.5), dissolved oxygen (O₂) (e.g.,between about 5-90% of air saturation), carbon dioxide (CO₂) (e.g.,between about 10-150 mmHg), agitation (between about 50 to 200 rpm) andhumidity, in addition to temperature (between about 30° C. to 37° C.).As an illustrative, yet nonlimiting, example, a suitable cell culturingmedium for the fed-batch processes of the present invention comprises achemically defined basal and feed medium, preferably one or bothcontaining the antioxidants of the invention (e.g., Example 1).

Animal cells, mammalian cells, cultured cells, animal or mammalian hostcells, host cells, recombinant cells, recombinant host cells, and thelike, are all terms for the cells that can be cultured according to theprocesses of this invention. Such cells are typically cell linesobtained or derived from mammals and are able to grow and survive whenplaced in either monolayer culture or suspension culture in mediumcontaining appropriate nutrients and/or growth factors. Growth factorsand nutrients that are necessary for the growth and maintenance ofparticular cell cultures are able to be readily determined empiricallyby those having skill in the pertinent art, such as is described, forexample, by Barnes and Sato, (1980, Cell, 22:649); in Mammalian CellCulture, Ed. J. P. Mather, Plenum Press, N Y, 1984; and in U.S. Pat. No.5,721,121.

Numerous types of cells can be cultured according to the methods of thepresent invention. The cells are typically animal or mammalian cellsthat can express and secrete, or that can be molecularly engineered toexpress and secrete, large quantities of a particular protein into theculture medium. It will be understood that the protein produced by ahost cell can be endogenous or homologous to the host cell.Alternatively, and preferably, the protein is heterologous, i.e.,foreign, to the host cell, for example, a human protein produced andsecreted by a Chinese hamster ovary (CHO) host cell.

Examples of mammalian proteins that can be advantageously produced bythe methods of this invention include, without limitation, cytokines,cytokine receptors, growth factors (e.g., EGF, HER-2, FGF-α, FGF-β,TGF-α, TGF-β, PDGF. IGF-1, IGF-2, NGF, NGF-β); growth factor receptors,including fusion or chimeric proteins. Other nonlimiting examplesinclude growth hormones (e.g., human growth hormone, bovine growthhormone); insulin (e.g., insulin A chain and insulin B chain),proinsulin; erythropoietin (EPO); colony stimulating factors (e.g.,G-CSF, GM-CSF, M-CSF); interleukins (e.g., IL-1 through IL-12); vascularendothelial growth factor (VEGF) and its receptor (VEGF-R); interferons(e.g., IFN-α, β, or γ); tumor necrosis factor (e.g., TNF-α and TNF-β)and their receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin;brain natriuretic peptide (BNP); clotting factors (e.g., Factor VIII,Factor IX, von Willebrands factor, and the like); anti-clotting factors;tissue plasminogen activator (TPA), e.g., urokinase or human urine ortissue type TPA; follicle stimulating hormone (FSH); luteinizing hormone(LH); calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.);CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bonemorphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.);neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF);neurotrophins, e.g., 3-6; renin; rheumatoid factor; RANTES; albumin;relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viralproteins or antigens; surface membrane proteins; ion channel proteins;enzymes; regulatory proteins; antibodies; immunomodulatory proteins,(e.g., HLA, MHC, the B7 family); homing receptors; transport proteins;superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs);neuromodulatory proteins; Alzheimer's Disease associated proteins andpeptides, (e.g., A-beta), and others as known in the art. Fusionproteins and polypeptides, chimeric proteins and polypeptides, as wellas fragments or portions, or mutants, variants, or analogs of any of theaforementioned proteins and polypeptides are also included among thesuitable proteins, polypeptides and peptides that can be produced by themethods of the present invention.

Nonlimiting examples of animal or mammalian host cells suitable forharboring, expressing, and producing proteins for subsequent isolationand/or purification include Chinese hamster ovary cells (CHO), such asCHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec.Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909;and WO 01/92337 A2), dihydrofolate reductase negative CHO cells(CHO/−DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA,77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121). Preferred areCHO cells, particularly, recombinant CHO cell lines established withDHFR or GS gene expression systems.

The cells suitable for culturing in the methods and processes of thepresent invention can contain introduced (e.g., via transformation,transfection, infection, or injection) expression vectors (constructs),such as plasmids and the like, that harbor coding sequences, or portionsthereof, encoding the proteins for expression and production in theculturing process. Such expression vectors contain the necessaryelements for the transcription and translation of the inserted codingsequence. Methods which are well known to and practiced by those skilledin the art can be used to construct expression vectors containingsequences encoding the produced proteins and polypeptides, as well asthe appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in J. Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubelet al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

Control elements, or regulatory sequences, are those non-translatedregions of the vector (e.g., enhancers, promoters, 5′ and 3′untranslated regions) that interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and host cellutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, can be used. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferred. The constructs for use in protein expressionsystems are designed to contain at least one promoter, an enhancersequence (optional, for mammalian expression systems), and othersequences as necessary or required for proper transcription andregulation of gene expression (e.g., transcriptional initiation andtermination sequences, origin of replication sites, polyadenylationsequences, e.g., the Bovine Growth Hormone (BGH) poly A sequence).

As will be appreciated by those skilled in the art, the selection of theappropriate vector, components for proper transcription, expression, andisolation of proteins produced in eukaryotic expression systems is knownand routinely determined and practiced by those having skill in the art.The expression of proteins by the cells cultured in accordance with themethods of this invention can be placed under the control of promoterssuch as viral promoters, e.g., cytomegalovirus (CMV), Rous sarcoma virus(RSV), phosphoglycerol kinase (PGK), thymidine kinase (TK), or theα-actin promoter. Further, regulated promoters confer inducibility byparticular compounds or molecules, e.g., the glucocorticoid responseelement (GRE) of mouse mammary tumor virus (MMTV) is induced byglucocorticoids (V. Chandler et al., 1983, Cell, 33:489-499). Also,tissue-specific promoters or regulatory elements can be used (G. Swiftet al., 1984, Cell, 38:639-646), if necessary or desired.

Expression constructs can be introduced into cells by a variety of genetransfer methods known to those skilled in the art, for example,conventional gene transfection methods, such as calcium phosphateco-precipitation, liposomal transfection, microinjection,electroporation, and infection or viral transduction. The choice of themethod is within the competence of the skilled practitioner in the art.It will be apparent to those skilled in the art that one or moreconstructs carrying DNA sequences for expression in cells can betransfected into the cells such that expression products aresubsequently produced in and/or obtained from the cells.

In a particular aspect, mammalian expression systems containingappropriate control and regulatory sequences are preferred for use inprotein expressing mammalian cells of the present invention. Commonlyused eukaryotic control sequences for generating mammalian expressionvectors include promoters and control sequences compatible withmammalian cells such as, for example, the cytomegalovirus (CMV) promoter(CDM8 vector) and avian sarcoma virus (ASV) πLN vector. Other commonlyused promoters include the early and late promoters from Simian Virus 40(SV40) (Fiers et al., 1973, Nature, 273:113), or other viral promoterssuch as those derived from polyoma, Adenovirus 2, and bovine papillomavirus. An inducible promoter, such as hMTII (Karin et al., 1982, Nature,299:797-802) can also be used.

Examples of expression vectors suitable for eukaryotic host cellsinclude, but are not limited to, vectors for mammalian host cells (e.g.,BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2,pRc/RSV, pSFV1 (Life Technologies); pVPakc Vectors, pCMV vectors, pSG5vectors (Stratagene), retroviral vectors (e.g., pFB vectors(Stratagene)), pcDNA-3 (Invitrogen), adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)), or modified forms of any of theforegoing. Vectors can also contain enhancer sequences upstream ordownstream of promoter region sequences for optimizing gene expression.

A selectable marker can also be used in a recombinant vector (e.g., aplasmid) to confer resistance to the cells harboring (preferably, havingstably integrated) the vector to allow their selection in appropriateselection medium. A number of selection systems can be used, includingbut not limited to, the Herpes Simplex Virus thymidine kinase (HSV TK),(Wigler et al., 1977, Cell, 11:223), hypoxanthine-guaninephosphoribosyltransferase (HGPRT), (Szybalska and Szybalski, 1992, Proc.Natl. Acad. Sci. USA, 48:202), and adenine phosphoribosyltransferase(Lowy et al., 1980, Cell, 22:817) genes, which can be employed in tk−,hgprt−, or aprt− cells (APRT), respectively.

Anti-metabolite resistance can also be used as the basis of selectionfor the following nonlimiting examples of marker genes: dhfr, whichconfers resistance to methotrexate (Wigler et al., 1980, Proc. Natl.Acad. Sci. USA, 77:357; and O'Hare et al., 1981, Proc. Natl. Acad. Sci.USA, 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072); neo,which confers resistance to the aminoglycoside G418 (Clinical Pharmacy,12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science,260:926-932; Anderson, 1993, Ann. Rev. Biochem., 62:191-21; May, 1993,TIB TECH, 11(5):155-215; and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene, 30:147). Methods commonly knownin the art of recombinant DNA technology can be routinely applied toselect the desired recombinant cell clones, and such methods aredescribed, for example, in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, N Y (1993); Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY; inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981.J. Mol. Biol., 150:1, which are incorporated by reference herein intheir entireties.

In addition, the expression levels of an expressed protein molecule canbe increased by vector amplification (for a review, see Bebbington andHentschel, “The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning”, Vol. 3,Academic Press, New York, 1987). When a marker in the vector systemexpressing a protein is amplifiable, an increase in the level ofinhibitor present in the host cell culture will increase the number ofcopies of the marker gene. Since the amplified region is associated withthe protein-encoding gene, production of the protein will concomitantlyincrease (Crouse et al., 1983, Mol. Cell. Biol., 3:257).

Vectors which harbor glutamine synthase (GS) or dihydrofolate reductase(DHFR) encoding nucleic acid as the selectable markers can be amplifiedin the presence of the drugs methionine sulphoximine or methotrexate,respectively. An advantage of glutamine synthase based vectors is theavailability of cell lines (e.g., the murine myeloma cell line, NSO)which are glutamine synthase negative. Glutamine synthase expressionsystems can also function in glutamine synthase expressing cells (e.g.,CHO cells) by providing additional inhibitor to prevent the functioningof the endogenous gene.

Vectors that express DHFR as the selectable marker include, but are notlimited to, the pSV2-dhfr plasmid (Subramani et al., Mol. Cell. Biol.1:854 (1981). Vectors that express glutamine synthase as the selectablemarker include, but are not limited to, the pEE6 expression vectordescribed in Stephens and Cockett, 1989, Nucl. Acids. Res., 17:7110. Aglutamine synthase expression system and components thereof are detailedin PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are incorporated by reference herein in theirentireties. In addition, glutamine synthase expression vectors that canbe used in accordance with the present invention are commerciallyavailable from suppliers, including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.).

Types of Cell Culture

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,continuous culture, batch culture and fed-batch culture. In a continuousculture, for example, fresh culture medium supplement (i.e., feedingmedium) is provided to the cells during the culturing period, while oldculture medium is removed daily and the product is harvested, forexample, daily or continuously. In continuous culture, feeding mediumcan be added daily and can be added continuously, i.e., as a drip orinfusion. For continuous culturing, the cells can remain in culture aslong as is desired, so long as the cells remain alive and theenvironmental and culturing conditions are maintained.

In batch culture, cells are initially cultured in medium and this mediumis neither removed, replaced, nor supplemented, i.e., the cells are not“fed” with new medium, during or before the end of the culturing run.The desired product is harvested at the end of the culturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run, i.e., the cells are“fed’ with new medium (“feeding medium”) during the culturing period.Fed-batch cultures can include the various feeding regimens and times,for example, daily, every other day, every two days, etc., more thanonce per day, or less than once per day, and so on. Further, fed-batchcultures can be fed continuously with feeding medium. The desiredproduct is then harvested at the end of the culturing/production run.

CHO Clones MDX1100 (Clone A)

In another embodiment, the CHO-K1 cells were transfected with a GS geneexpression system in order to establish a stable cell line expressing ahuman IgG4 antibody.

The transfected and cloned cells expressing the antibody was grown inmedia with varied concentrations of the tested factors according to themethods of the invention. [see example 2 to 5]

The antibody produced by clone A is described in U.S. application2005/0191293, which is incorporated herein by reference.

Myostatin (Clone G)

In another embodiment, the dhfr− negative Chinese Hamster Ovary (CHO)cell line DG44 was transfected in order to establish a stable cell lineexpressing a recombinant human fusion protein.

The transfected and cloned CHO DG44 cells expressing the fusion proteinwas grown in media with varied concentrations of the tested factorsaccording to the methods of the invention. [see examples 2 to 5]

The fusion protein produced by clone G is described in U.S. Pat. No.8,933,199, which is incorporated herein by reference.

aCD40L (Clone B)

In another embodiment, the dhfr− negative Chinese Hamster Ovary (CHO)cell line DG44 was transfected in order to establish a stable cell lineexpressing a recombinant human fusion protein.

The transfected and cloned CHO DG44 cells expressing the antibody wasgrown in media with varied concentrations of the tested factorsaccording to the methods of the invention. [see examples 2 to 5]

The antibody produced by clone B is described in U.S. Pat. No.8,895,010, which is incorporated herein by reference.

aLag 3 (Clone C)

In another embodiment, the dhfr− negative Chinese Hamster Ovary (CHO)cell line DG44 was transfected in order to establish a stable cell lineexpressing a human IgG4 antibody.

The transfected and cloned CHO DG44 cells expressing the antibody wasgrown in media with varied concentrations of the tested factorsaccording to the methods of the invention. [see Examples 2 to 5]

The antibody produced by clone C is described in PCT applicationWO2014/008218, which is incorporated herein by reference.

MDX1110 (Clone D)

In another embodiment, the dhfr− negative Chinese Hamster Ovary (CHO)cell line CHO-Ms704 were transfected in order to establish a stable cellline expressing a human IgG4 antibody.

The transfected and cloned cells expressing the antibody was grown inmedia with varied concentrations of the tested factors according to themethods of the invention. [see examples 2 to 5]

The antibody produced by clone D is described in U.S. Pat. No.8,383,118, which is incorporated herein by reference.

aKir (Clone F)

In another embodiment, the CHO-K1 cells were transfected with a GS geneexpression system in order to establish a stable cell line expressing ahuman IgG4 antibody.

The transfected and cloned cells expressing the antibody was grown inmedia with varied concentrations of the tested factors according to themethods of the invention. [see examples 2 to 5]

The antibody produced by clone F is described in PCT applicationWO2008/084106, which is incorporated herein by reference.

Pharmaceutical Formulations

In certain embodiments, produced polypeptides will have pharmacologicactivity and will be useful in the preparation of pharmaceuticals.Inventive compositions as described above may be administered to asubject or may first be formulated for delivery by any available routeincluding, but not limited to parenteral, intravenous, intramuscular,intradermal, subcutaneous, oral, buccal, sublingual, nasal, bronchial,opthalmic, transdermal (topical), transmucosal, rectal, and vaginalroutes. Inventive pharmaceutical compositions typically include apurified polypeptide expressed from a mammalian cell line, a deliveryagent in combination with a pharmaceutically acceptable carrier. As usedherein, the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into compositions of the presentinvention. For example, a polypeptide produced according to the presentinvention may be conjugated to drugs for systemic pharmacotherapy, suchas toxins, low-molecular-weight cytotoxic drugs, biological responsemodifiers, and radionuclides (see e.g., Kunz et al., Calicheamicinderivative-carrier conjugates, US20040082764 A1). Additional ingredientsuseful in preparing pharmaceutical compositions in accordance with thepresent invention include, for example, flavoring agents, lubricants,solubilizers, suspending agents, fillers, glidants, compression aids,binders, tablet-disintegrating agents, encapsulating materials,emulsifiers, buffers, preservatives, sweeteners, thickening agents,coloring agents, viscosity regulators, stabilizers or osmo-regulators,or combinations thereof.

Alternatively or additionally, a polypeptide produced according to thepresent invention may be administered in combination with (whethersimultaneously or sequentially) one or more additional pharmaceuticallyactive agents. An exemplary list of these pharmaceutically active agentscan be found in the Physicians' Desk Reference, 55 Edition, published byMedical Economics Co., Inc., Montvale, N.J., 2001, incorporated hereinby reference in its entirety. For many of these listed agents,pharmaceutically effective dosages and regimens are known in the art;many are presented in the Physicians' Desk Reference itself.

Solid pharmaceutical compositions may contain one or more solidcarriers, and optionally one or more other additives such as flavoringagents, lubricants, solubilizers, suspending agents, fillers, glidants,compression aids, binders or tablet-disintegrating agents or anencapsulating material, suitable solid carriers include, for example,calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin,starch, gelatin, cellulose, methyl cellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low melting waxes or ion exchangeresins, or combinations thereof. In powder pharmaceutical compositions,the carrier may be a finely divided solid which is in admixture with thefinely divided active ingredient. In tablets, the active ingredient isgenerally mixed with a carrier having the necessary compressionproperties in suitable proportions, and optionally, other additives, andcompacted into the desired shape and size.

Liquid pharmaceutical compositions may contain the polypeptide expressedaccording to the present invention and one or more liquid carriers toform solutions, suspensions, emulsions, syrups, elixirs, or pressurizedcompositions. Pharmaceutically acceptable liquid carriers include, forexample water, organic solvents, pharmaceutically acceptable oils orfat, or combinations thereof. The liquid carrier can contain othersuitable pharmaceutical additives such as solubilizers, emulsifiers,buffers, preservatives, sweeteners, flavoring agents, suspending agents,thickening agents, colors, viscosity regulators, stabilizers orosmo-regulators, or combinations thereof. If the liquid formulation isintended for pediatric use, it is generally desirable to avoid inclusionof, or limit the amount of, alcohol.

Examples of liquid carriers suitable for oral or parenteraladministration include water (optionally containing additives such ascellulose derivatives such as sodium carboxymethyl cellulose), alcoholsor their derivatives (including monohydric alcohols or polyhydricalcohols such as glycols) or oils (e.g., fractionated coconut oil andarachis oil). For parenteral administration the carrier can also be anoily ester such as ethyl oleate and isopropyl myristate. The liquidcarrier for pressurized compositions can be halogenated hydrocarbons orother pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be administered parenterally, for example by,intramuscular, intraperitoneal, epidural, intrathecal, intravenous orsubcutaneous injection. Pharmaceutical compositions for oral ortransmucosal administration may be either in liquid or solid compositionform.

In certain embodiments, a pharmaceutical composition is formulated to becompatible with its intended route of administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition should be sterile and should befluid to the extent that easy syringability exists. Advantageously,certain pharmaceutical formulations are stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. In general, therelevant carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Incertain cases, it will be useful to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating thepurified polypeptide in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the purified polypeptide expressed from a mammaliancell line into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, advantageous methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, thepurified polypeptide can be incorporated with excipients and used in theform of tablets, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared using a fluid carrier, e.g., for useas a mouthwash. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Such preparationsmay be mixed chewable or liquid formulations or food materials orliquids if desirable, for example to facilitate administration tochildren, to individuals whose ability to swallow tablets iscompromised, or to animals. Formulations for oral delivery mayadvantageously incorporate agents to improve stability within thegastrointestinal tract and/or to enhance absorption.

Alternatively, the compounds can be administered in the form of asuppository or pessary, or they may be applied topically in the form ofa gel, hydrogel, lotion or other glycerides, solution, cream, ointmentor dusting powder.

In some embodiments, compositions are prepared with carriers that willprotect the polypeptide against rapid elimination from the body, such asa controlled release formulation, including implants andmicroencapsulated delivery systems. In general, inventive compositionsmay be formulated for immediate, delayed, modified, sustained, pulsed,or controlled-release delivery. Biodegradable, biocompatible polymerscan be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Suitable materials can also be obtained commerciallyfrom Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to infected cells withmonoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

In some embodiments, pharmaceutical compositions of the presentinvention are provided in unit dosage form, such as tablets or capsules.It may be advantageous to formulate oral or parenteral compositions inunit dosage form for ease of administration and uniformity of dosage. Insuch form, the composition is sub-divided in unit dose containingappropriate quantities of the polypeptide. The unit dosage forms can bepackaged compositions, for example packeted powders, vials, ampoules,pre-filled syringes or sachets containing liquids. As one skilled in theart will recognize, therapeutically effective unit dosage will depend onseveral factors, including, for example, the method of administration,the potency of the polypeptide, and/or the weight of the recipient andthe identities of other components in the pharmaceutical composition.

A polypeptide expressed according to the present invention can beadministered at various intervals and over different periods of time asrequired, e.g., daily, weekly, biweekly, monthly, etc. The skilledartisan will appreciate that certain factors can influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Treatment of a subject with a polypeptide as described hereinmay comprise a single treatment or a series of treatments. It isfurthermore understood that appropriate doses may depend upon thepotency of the polypeptide and may optionally be tailored to theparticular recipient, for example, through administration of increasingdoses until a preselected desired response is achieved. It is understoodthat the specific dose level for any particular animal subject maydepend upon a variety of factors including the activity of the specificpolypeptide employed, the age, body weight, general health, gender, anddiet of the subject, the time of administration, the route ofadministration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Inventive pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

Example 1

Experiment Procedures: a vial of frozen cells were thawed into BMSproprietary basal medium 17I with or without growth factors (see detailsfor each individual experiment) and cultured for 3 passages. The cellswere then transferred to modified 17I prototype media with differenttreatments with increased or decreased concentration of nutrientcomponents (see details for each individual experiment). The cells wereadapted in the prototype media for 3 passages prior to setting up batchcultures in order to evaluate the performance of the prototype media. Ondays 12 of the fed-batch culture, supernatant samples were harvested andpurified with proA Chromatography columns. The purified protein sampleswere then subjected to protein quality attribute analyses (see FIG. 1 ).Cell culture parameters: cells were cultured either in 50 ml spin tubeswith an initial volume of 25 ml and a shaking speed of 300 rpm or 500 mlshaker flasks with an initial volume of 150 ml and a shaking speed of150 rpm on an orbital shaker with 25 mm throw distance. Temperature forthe culture was controlled at 37° C. and CO2 was controlled at 6%. Cellswere cultured in spin tubes for Experiment 2, and in shaker flasks forthe rest of the experiments.

High molecule weight analyses: high molecular weight species wereanalyzed with size exclusion HPLC with a Tosoh Bioscience TSKgelSuperSW3000 column and SuperSW guard column, and an aqueous bufferedmobile phase.

Glycosylation profile analyses: intact mass analysis with LC-MS was usedto compare the glycosylation profiles of protein samples. About 0.5 μgof each protein sample was loaded onto a Poros reversed phase 2.1×100 mmcolumn (Applied Biosystems, Foster City, Calif.) equilibrated with 0.1%formic acid in 20% (v/v) acetonitrile and separated by a gradientelution from 20% to 50% acetonitrile with 0.1% formic acid in 25 min, ata flow rate of 0.25 mL/min. Eluent was electrosprayed into a Q-ToFUltima mass spectrometer (Waters, Milford, Mass.) for online detection.The mass spectra were combined and deconvoluted using MaxEnt1 algorithm(Waters, Milford, Mass.). Peaks were assigned at 100 ppm mass accuracy.

Statistical Analysis: Customized factorial (fractional or responsesurface depending on the levels studied for each factor) Design ofExperiment (DOE) was used to generate the run conditions usingstatistical software JMP® 10.0.0 (SAS Institute Inc.). Each response(e.g., HMW %, Gal %, etc) was analyzed using the stepwise regressionmethod with minimum AICc, minimum BIC, or p-value threshold stoppingrules. Only when p-values were less than 0.05 were the terms selected togenerate a mathematical model with standard least squares method. Themodel was then used to analyze the impact of the factors on theresponses and to predict the max or min value for each response.

Example 2

Basal medium 17I was modified with the components listed in Table 1.Except for the lipids group, the concentration for each component wasshown as a percentage increase or decrease to the existing concentrationfor the component in 17I. The specific component and concentration forthe lipids group is proprietary to Hyclone. The modified components weregrouped into amino acids, vitamins, lipids, antioxidants, and phosphatebased on the functions or properties for the chemical compounds. Thegroups were then the study factors used to generate prototype mediaconditions shown in table 12 using a response surface statistical designapproach. Cells were cultured in the prototype media without any growthfactors following the protocol described in Example 1 above. Inaddition, cells were also cultured in prototype medium #9 with a growthfactor (1 mg/L insulin) and served as a control to compare the impact ofgrowth factors on the protein quality attributes.

TABLE 12 Run # AAs Vitamins Lipids Antioxidants Phosphate 1 0 0 0 1 0 21 1 1 −1 0 3 0 0 0 0 0 4 1 0 1 1 −1 5 1 −1 −1 −1 1 6 −1 1 −1 1 −1 7 0 00 0 0 8 0 0 1 0 1 9 −1 0 −1 −1 0 10 1 0 −1 0 −1 11 −1 −1 0 0 −1 12 0 −1−1 1 0 13 −1 0 0 −1 1 14 1 −1 0 0 0 15 −1 −1 1 1 1 16 −1 1 1 0 0 17 0 1−1 0 1 18 0 −1 1 −1 −1 19 0 1 0 −1 −1 20 1 1 0 1 1

Example 3

Experiment Description: Following the cell culture protocol described inExample 1 above, cells were cultured in 17I with different concentrationof phosphate ranging from 0.5 to 1.4 g/L either in the presence orabsence of growth factors (1 mg/L insulin) as described in Table 13below.

TABLE 13 Clone Condition Medium Phosphate (g/L) B GF-Free 17I 0.5 0.81.1 1.4 GF 17I 0.5 0.8 1.1 1.4 G GF-Free 17I 0.5 0.8 1.1 1.4 GF 17I 0.50.8 1.1 1.4

Example 4

Experiment Description: The initial media for this experiment was basalmedium 17I with additional amino acids, antioxidants, and phosphate atlevel “+1” (see Table 1). The medium was then modified for the singlecomponent or group of components listed in Table 14 below.

TABLE 14 Level 1 Level 2 Level 3 Concentration Group Components (Coded)(Coded) (Coded) Range Zinc Zinc 0 (−1) +50% (0) +100% (+1) 0.01-100 mg/LFeSO4 FeSO4 0 (0) −50% (−1) −70% (−2) 0.01-100 mg/L Iron Carrier E N/A0.01-1 g/L IC1 0.01-1 g/L IC2 0.01-1 g/L None N/A Vitamins Folic Acid(B9) 0 (0) −50% (−1) NA 0.01-100 mg/L Cyanochobalamin 0 (0) −50% (−1) NA0.01-100 mg/L (B12) Riboflavin (B2) 0 (0) −50% (−1) NA 0.01-100 mg/L L-L-Asparagine 0 (−1) +50% (0) +100% (+1) 0.1-2 g/L Asparagine

The groups were then the study factors used to generate prototype mediaconditions shown in Table 15 using a customized statistical Design ofExperiment (DoE) approach. Cells were cultured in the prototype mediawithout any growth factors following the protocol described in Example 1above.

TABLE 15 Iron Run # Zinc Asn FeSO4 Carrier Colorant 1 −1 0 0 E 0 2 1 0−1 E 0 3 0 0 −2 E 0 4 1 1 0 none 0 5 −1 1 −2 none 0 6 1 −1 0 IC1 0 7 0 0−1 IC1 0 8 −1 −1 −2 IC1 0 9 0 0 0 IC2 0 10 −1 0 −1 IC2 0 11 −1 −1 0 none−1 12 1 0 0 E −1 13 −1 0 −1 E −1 14 1 0 −2 IC2 −1 15 0 0 −1 none −1 16 1−1 −2 none −1 17 −1 1 0 IC1 −1 18 1 1 −2 IC1 −1 19 1 0 −1 IC2 −1 20 −1 0−2 IC2 −1 E: EDTA; none: no Fe Carrier; IC1 & IC2: iron carrierproprietary to Hyclone at concentration 1 & 2

Example 5

Experiment Description: The initial media for this experiment was basalmedium 17I with additional amino acids, antioxidants, and phosphate atlevel “+1” (see table 1). The medium was then modified for the singlecomponent or group of components listed in table 16 below.

TABLE 16 Level 1 Level 2 Level 3 Concentration Group Components (Coded)(Coded) (Coded) Range Zinc Zinc 0 (0) +50% (+1) NA 0.01-100 mg/L FeSO4FeSO4 −30% (−1) 0 (0) +50% (+1) 0.01-100 mg/L Iron Carrier E N/A 0.01-1g/L IC1 0.01-1 g/L Sodium Citrate (SC) 0.01-1 g/L None N/A L-AsparticL-Aspartic Acid 0 (−1) +50% (0) +100% (+1) 0.1-2 g/L Acid L-AsparagineL-Asparagine 0 (−1) +50% (0) +100% (+1) 0.1-2 g/L

The groups were then the study factors used to generate prototype mediaconditions shown in Table 17 using a customized statistical Design ofExperiment (DoE) approach. Cells were cultured in the prototype mediawithout any growth factors following a protocol described in Example 1above. E: EDTA; none: no Fe Carrier; IC1: iron carrier proprietary toHyclone; SC: sodium citriate.

TABLE 17 Run # Zinc Carrier Asp Asn FeSO4 1 1 IC1 −1 0 −1 2 0 None −1 −10 3 1 SC −1 0 0 4 1 SC 1 −1 1 5 0 None 1 1 0 6 0 E 0 1 −1 7 1 E 0 −1 −18 0 E 0 −1 1 9 0 SC 0 1 1 10 0 None 0 −1 1 11 1 IC1 1 0 1 12 1 SC 1 1 −113 0 IC1 0 1 0 14 1 None 0 0 −1 15 0 IC1 1 −1 −1 16 1 E 0 1 1 17 0 SC 0−1 −1 18 0 IC1 −1 −1 1 19 0 None −1 −1 −1 20 1 None 0 0 1

What is claimed is:
 1. A method of increasing sialic acid content of arecombinant glycoprotein expressed in Chinese Hamster Ovary (CHO) cellsunder suitable cell culture conditions comprising: adding at least 100mg/ml of zinc, about 0.1 g/L aspartic acid, about 0.1 g/L phosphate tothe cell culture medium, wherein the cell culture medium is maintainedunder following conditions: a) pH between 6.5-7.5; b) dissolved oxygenbetween 5% and 90% of air saturation; c) carbon dioxide between 10 mmHgand 150 mmHg; and d) temperature between 30° C. and 37° C.; and whereinthe sialic content of the glycoprotein is increased.
 2. The method ofclaim 1, wherein the cell culture medium is chemically defined.
 3. Themethod of claim 2, wherein the chemically defined cell culture medium isbasal medium.
 4. The method of claim 3, wherein the chemically definedbasal medium does not contain added serum or hydrolysates.
 5. The methodof claim 3, wherein the chemically defined basal medium is protein-free.6. The process according to claim 1, wherein the glycoprotein is arecombinant antibody, antibody fragment or fusion protein.